Sructural study of immuno regulatory proteins by NMR spectroscopy

作为逃避宿主免疫系统检测的策略的一部分，小鼠巨细胞病毒（MCMV）编码三种调节细胞表面主要组织相容性复合体I类（MHC-I）分子表达的蛋白：MHC-I同源物m152/gp40，以及m02-m16家族成员m04/gp34和m06/gp48。先前对m04蛋白的研究揭示了m02-m16家族免疫球蛋白独有的发散性免疫球蛋白（Ig）样折叠。在这里，我们设计和表征重组m06，并通过几种技术研究其与MHC-I分子H2-Ld的全长和截短形式的相互作用。此外，我们利用截断的H2-Ld， mini-H2-Ld，仅由12个平台结构域组成，利用溶液核磁共振绘制了m06蛋白在MHC-I上的相互作用足迹。Mini-H2-Ld与高亲和肽在体外重新折叠，产生的分子显示出出色的核磁共振光谱特征，允许完整的骨架分配。这些基于核磁共振的研究表明，m06与位于肽结合平台下的一个离散位点紧密结合，该位点与MHC-I重链上的2-微球蛋白（2m）界面部分重叠，这与体外结合实验一致，表明m06与2m相关的MHC-I之间的复合物形成显著减少。此外，我们进行了核磁共振弛豫实验来表征自由的mini-H2-Ld MHC-I分子的ps-ns动力学，揭示了相互作用的位置是高度有序的。这项研究揭示了m06与MHC-I相互作用的机制，表明在多肽装载途径的早期阶段，靶MHC-I分子的结构操纵。

As part of its strategy to evade detection by the host immune system, murine cytomegalovirus (MCMV) encodes three proteins that modulate cell surface expression of major histocompatibility complex class I (MHC-I) molecules: the MHC-I homolog m152/gp40, as well as the m02-m16 family members m04/gp34 and m06/gp48. Previous studies of the m04 protein revealed a divergent immunoglobulin (Ig)-like fold that is unique to immunoevasins of the m02-m16 family. Here, we engineer and characterize recombinant m06 and investigate its interactions with full length and truncated forms of the MHC-I molecule H2-Ld by several techniques. Furthermore, we employ solution NMR to map the interaction footprint of the m06 protein on MHC-I, taking advantage of a truncated H2-Ld, mini-H2-Ld, consisting of only the 12 platform domain. Mini-H2-Ld refolded in vitro with a high-affinity peptide to yields a molecule that shows outstanding NMR spectral features, permitting complete backbone assignments. These NMR-based studies reveal that m06 binds tightly to a discrete site located under the peptide-binding platform that partially overlaps with the 2-microglobulin (2m) interface on the MHC-I heavy chain, consistent with in vitro binding experiments showing significantly reduced complex formation between m06 and 2m-associated MHC-I. Moreover, we carry out NMR relaxation experiments to characterize the ps-ns dynamics of the free mini-H2-Ld MHC-I molecule, revealing that the site of interaction is highly ordered. This study provides insight into the mechanism of the interaction of m06 with MHC-I, suggesting a structural manipulation of the target MHC-I molecule at an early stage of the peptide-loading pathway.